Read-through compound prodrugs suppressing premature nonsense mutations

ABSTRACT

Premature termination codon readthrough prodrug compounds, compositions thereof, and methods of making and using the same are provided. In certain embodiments, the compounds are of Formula Ia or a pharmaceutically acceptable salt, solvate, polymorph, hydrate, ester, isomer, stereoisomer, or tautomer thereof, 
     
       
         
         
             
             
         
       
     
     wherein R, A and W are as described herein.

PRIOR RELATED APPLICATION

This application claims the benefit of, and priority to, U.S.Provisional Application No. 61/928,334, entitled “READ-THROUGH COMPOUNDPRODRUGS SUPPRESSING PREMATURE NONSENSE MUTATION,” filed Jan. 16, 2014,which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.NS076761, awarded by the National Institutes of Health and Grant No.W81XWH-13-1-0207, awarded by the United States Army Medical Research andMateriel Command. The Government has certain rights in this invention.

FIELD

Provided herein are compounds capable of targeting nonsense mutationsand therefore allowing read-through of premature termination codons. Incertain embodiments, the prodrugs are conjugates of compound 5 or itspharmaceutically acceptable salts, isomers, complexes, polymorphs,hydrates or esters thereof. The prodrugs are capable of conversion bynatural biological processes into an active ingredient (for example,compound 5) which can suppress early termination of protein synthesisand therefore can treat genetic diseases characterized by nonsensemutations.

Also provided herein are pharmaceutical formulations of the compounds,methods of using the compounds to treat patients afflicted with geneticdisorders characterized by nonsense mutations, and methods ofsynthesizing the compounds.

BACKGROUND

Translation termination is signaled by three stop codons: UAA, UAG, andUGA. This mechanism is highly conserved, although each stop codon has adifferent efficiency for terminating translation. UGA is considered tobe a “leaky” stop codon with the highest intrinsic readthroughpotential. UAA shows high fidelity and little intrinsic readthroughpotential, whereas UAG has intermediate fidelity (see, e.g., Weiner andWeber, 1973, J. Mol. Biol. 80:837-855). Nonsense mutations createprimary premature termination codons (PTCs) and result in either noformation of the target protein or truncated protein with impairedstability. Large numbers of genetic disorders are caused by nonsensemutations for Which compound-induced readthrough of prematuretermination codons (PTCs) can be exploited as a potential treatmentstrategy.

Certain compounds influence the fidelity of stop codon recognition andinduce readthrough of primary PTCs, which allows translation of somefull-length protein. In many cases, the readthrough-induced protein isfunctional, even when it contains a wrongly incorporated amino acid(Keeling and Bedwell, 2005, Curr. Pharmacogenetics 3:259-269; Zingman etal., 2007, Clin. Pharmacol. Ther. 81:99-103).

It is estimated that 30% of human disease-causing alleles are nonsensemutations (Du et al., 2009, J. Exp. Med., 206 (10): 2285). Other typesof mutations, such as frameshift and splicing mutations, lead tosecondary PTCs; however, these are not therapeutic targets forreadthrough compounds (RTCs). Considering that >1,800 distinct geneticdisorders are caused by nonsense mutations, the readthrough of primaryPTCs has treatment potential for large numbers of patients.

To date, most reported PTC-RTCs that are active in mammalian cells havebelonged to the aminoglycoside antibiotics class (Keeling and Bedwell,2005, Curr. Pharmacogenomics, 3:259-269; Zingman et al., 2007, Clin.Pharmacol. Ther., 81:99-103). Certain types of aminoglycosides caninduce ribosomes to read through PTC mutations via insertion of a randomamino acid by near-cognate transfer RNA. The therapeutic potential ofaminoglycosides has been evaluated in the laboratory for differentgenetic models, such as cystic fibrosis (see, e.g., Du et al., 2002, J.Mol. Med. 80:595-604), muscular dystrophy (see, e.g., Loufrani et al.,2004, Arterioscler. Thromb. Vasc. Biol. 24:671-676), Hurler syndrome(Keeling et al., 2001, Hum. Mol. Genet. 10:291-299), cystinosis(Helip-Wooley et al., 2002, Mol. Genet. Metab. 75:128-133), spinalmuscular atrophy (Sossi et al., 2001, Eur. J. Hum. Genet. 9:113-120),ataxia-telangiectasia (Lai et al., 2004, Proc. Natl. Acad. Sci. USA.101:15676-15681), and type 1 Usher syndrome (Rebibo-Sabbah et al., 2007,Hum. Genet. 122:373-381). Clinical trials also indicate thataminoglycosides can induce some functional protein production; however,the therapeutic benefits remain uncertain (see, e.g., Politano et al.,2003, Acta Myol. 22:15-21). Furthermore, the toxicity of most commercialaminoglycosides in mammals has greatly diminished their potential forsuccessful readthrough therapy (Mingeot-Leclercq and Tulkens, 1999,Antimicrob. Agents Chemother. 43:1003-1012; Guan et al., 2000, Hum. Mol.Genet. 9:1787-1793). Therefore, efforts are underway to develop betteraminoglycoside derivatives with reduced toxicity and enhanced activity(Nudelman et al., 2006, Bioorg. Med. Chem. Lett. 16:6310-6315;Rebibo-Sabbah et al., 2007, Hum. Genet. 122:373-381).

Recently, PTC Therapeutics (South Plainfield, N.J.) described a moreefficient non-aminoglycoside RTC, PTC124, which was developedsynthetically by screening >800,000 chemicals and analogues using aluciferase-based high-throughput screening (HTS) assay (see, e.g., Welchet al., 2007, Nature. 447:87-91). A phase-I clinical study in cysticfibrosis confirmed that PTC124 is generally well tolerated and appearsto have more efficient readthrough activity than aminoglycosides(Hirawat et al., 2007, J. Clin. Pharmacol. 47:430-444). Moreover, PTC124does not induce ribosomal readthrough of normal stop codons. A phase-IIclinical trial is underway (Kerem et al., 2008, Lancet. 372:719-727).However, a recent study indicates that the initial discovery of PTC124by HTS can have been biased by its direct effect on the FLuc (fireflyluciferase) reporter used (Auld et al., 2009, Proc. Natl. Acad. Sci.USA. 106:3585-3590), indicating the importance of aluciferase-independent HTS assay for future drug screening.

Recent findings have demonstrated that the specificity of the HTSutilized for the identification of the read-through compound may havebeen compromised by the ligand-induced stabilization of the reporterprotein used for the screen (Auld, et al., (2009), Proc. Natl. Acad.Sci. 106, 3585-3590). Although other interpretations have been offered(Peltz et al., P (2009), Proc. Natl. Acad. Sci. USA, 106, E64), strongevidence have been presented in support of the post-translationalactivity of PTC124 (Auld et al., 2010, Proc. Natl. Acad. Sci. USA, 107,4878-4883; Thorne et al., 2010, Chem. Biol., 17, 646-657). Despite theoff-target effects, PTC124 has been shown to suppress nonsense mutationsin different disease models and has reached clinical testing in patients(Sermet-Gaudelus et al., 2010, Am. J. Respir. Crit. Care Med., Vol. 182,No. 10, pp. 1262-1272; Welch et al., 2007, Nature. 447, 87-91). However,the indeterminate efficacy of Ataluren (PTC124) in clinical trials forDuchenne boys supports the need of identifying new drugs that could beused to suppress nonsense mutations in the clinical scenario.

Recently, a sensitive and quantitative high-throughput screening methodwas developed by Du et al. (Du et al., 2009, J. Exp. Med. 206,2285-2297) and used to screen low-molecular mass non-aminoglycosidecompound libraries to identify potential drugs with PTC read-throughactivity. The screening protocol involved the use of a proteintranscription/translation (PTT)—enzyme-linked immunosorbent assay(ELISA), using ataxia-telangiectasia (A-T) as a genetic disease model.This PTT-ELISA was driven by plasmid templates containing prototypic ATMmutations, patterned after specific disease-causing A-T (Du et al.,2009, J. Exp. Med. 206, 2285-2297). The screen of nearly 34,000compounds led to the identification of compound 5 (provided in FIG. 1).This compound was shown to have biological activity in differentlymphoblastoid cell lines derived from A-T patients containing each ofthe three types of nonsense mutations (TGA>TAA>TAG). Furthermore,compound 5 restored full-length dystrophin in mdx cells in culture (Duet al., 2009, J. Exp. Med. 206, 2285-2297). Altogether, these datademonstrated that the compound has read-through activity on differenttypes of proteins, in more than one species and cell lineage, and thatactivity is independent of the location of the premature stop codonwithin the transcript. Furthermore, compound 5 was shown to notread-through normal termination codons, thus confirming specificity forPTCs (Du et al., 2009, J. Exp. Med. 206, 2285-2297).

We have recently demonstrated that systemic administration of compound 5restores functional levels of dystrophin expression in skeletal musclesof the mdx mice (Kayali et al., 2012, Hum. Mol. Genet., 21, 4007-4020):

Dystrophin protein was detected in all muscle groups analyzed, includingdiaphragm and heart, two of the muscles most difficult muscle to targetby any of the therapeutic approaches currently being developed.Dystrophin was significantly higher than that achieved by PTC124 andresulted in a significant increase in muscle strength over mice that didnot receive the RTCs. The improvement in muscle function was paralleledby a decrease in creatine kinase (CK) levels a marker of muscledegeneration. These data demonstrate that compound 5 is a valid drug forthe treatment of Duchenne muscular dystrophy (DMD) and suggest that manyother disorders can benefit from its successful development into a drug.

However, one of the major limitations in further developing compound 5for clinical applications is its low solubility.

SUMMARY

A successful commercialization of compound 5 is likely to require theoptimization of a structure of compound 5 with improved solubility andoral viability as it is likely that oral administration is the bestroute to deliver the compound to target organs and at doses necessary toachieve an effect. Therefore, provided herein are compounds,compositions comprising the compounds, methods of making the compoundsand compositions, and methods of using the compounds for treating orameliorating a medical condition associated with premature terminationcodons (PTCs) in RNA. In certain embodiments the compounds providedherein have superior solubility, efficacy, and/or bioavailability fortreating or ameliorating a medical condition associated with prematuretermination codons (PTCs) in RNA.

In certain embodiments, provided herein is a compound according toFormula Ia:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, wherein:

W is —NR^(a)R^(b), —C(O)OR⁴, —C(O)NR^(a)R^(b), or -HetAr;

A is a bond from C(O) to W, —(CH₂)_(f)CH(R¹)(CH₂)_(g)—,—(CH₂)_(f)C(R^(a)R^(b))(CH₂)_(g)—, —(CH₂CH₂O)_(h)(CH₂)_(t)—,—(CH₂)_(t)(OCH₂CH₂)_(h)—, —(CH₂)_(t)N(R^(e))CH₂CH₂Z, or

or, in the alternative, A and W combine to form

R¹ is —H, —(CH₂)_(n)CH₃, —CH(CH₃)CH₂CH₃, —CF₃, —CH₂(Ar), —CH₂(HetAr),—CH₂S(O)_(m)CH₃, —CH₂CH₂S(O)_(m)CH₃, —CH₂(CH₂)_(n)NR^(e)R^(d), —CH₂OH,—CH(CH₃)OH, or —(CH₂)_(t)COOH;

R² is —H, —CH₃, —OH, or —CF₃;

each of R^(a) and R^(b) is independently R^(e); or, in the alternative,R^(a) and R^(b), together with the nitrogen or carbon atom to which theyare attached, combine to form a 4 to 7-membered ring heterocyclic ringwhich optionally contains additional heteroatoms selected from O,NR^(g), and S(O)_(m);

R^(c) is —H, —CH₃, —(CH₂)_(n)CH₃, —CH(R²)CH₃, —CH₂-pyridyl, orCH₂-imidazolyl;

R^(d) is —H, —CH₃, or —(CH₂)_(n)CH₃; or, in the alternative, R^(c) andR^(d), together with the nitrogen atom to which they are attached, forma 4-7 membered heterocyclic ring which optionally contains additionalheteroatoms selected from O, NR^(e), and S(O)_(m);

each R^(e) is independently —H, —(CH₂)_(n)CH₃, —CH(CH₃)₂,—CH(CH₃)CH₂CH₃, —(CH₂CH₂O)_(p)R³, or CH₂HetAr; each R^(g) isindependently —H, —(CH₂)_(n)CH₃, —CH(CH₃)₂, —CH(CH₃)CH₂CH₃,—(CH₂CH₂O)_(p)R³, —CH₂-phenyl or —CH₂-phenyl optionally substituted withF, Cl, —CH₃, —OCH₃, —OCF₃, or HetAr;

each R³ is independently —H, —CH₃, —OH, or —CF₃; alternatively, each R³is independently —H, —CH₃, —CH₂CH₂—OH, or —CF₃;

each R⁴ is independently —H or —(CH₂)_(n)NR^(c)R^(d);

each HetAr is independently a heteroaryl group optionally selected frompyridyl, pyrimidyl, C-imidazolyl and N-imidazolyl;

D is CH or N;

Q is —O—, —NR^(a)—, —S(O)_(m)—, or —CH—W—;

each k is independently 1, 2, 3 or 4;

each u is independently 1, 2 or 3;

each v is independently 1, 2 or 3;

each p is independently 1 or 2;

each f is independently 0, 1 or 2;

each g is independently 0, 1 or 2;

each h is independently 1 or 2;

each n is independently 0, 1, 2, 3, or 4;

each m is independently 0, 1 or 2;

each of q₁ and q₂ is independently 0, 1, 2 or 3;

each s is independently 0, 1, 2 or 3; and

each t is independently 0, 1, 2 or 3.

In certain embodiments provided herein is a composition. The compositioncomprises at least one compound or a pharmaceutically acceptable salt orprodrug thereof in an amount effective for treating or ameliorating amedical condition associated with premature termination codons (PTCs) inRNA, as described herein.

In some embodiments of the composition, the composition comprises twocompounds, each of the two compounds described herein.

In some embodiments of the compositions provided herein, the compositionfurther comprises a pharmaceutically acceptable carrier.

In some embodiments of the compositions provided herein, the compositionis formulated in a formulation for local or systemic delivery. Examplesof such formulations are formulations for oral administration,injection, topical administration, pulmonary administration, or implant.

In still a further aspect, it is provided a method. The method comprisesproviding a compound having the ability to read through prematuretermination codons (PTCs) in RNA, and forming a composition comprisingthe compound, a pharmaceutically acceptable salt thereof, or a prodrugthereof. The compound is described herein.

In some embodiments of the method, the composition comprises twocompounds, each of the two compounds described herein.

In some embodiments of the methods described herein, the compositionfurther comprises a pharmaceutically acceptable carrier.

In some embodiments of the methods described herein, the composition isformulated in a formulation for local or systemic delivery. Examples ofsuch formulations are formulations for oral administration, injection,topical administration, pulmonary administration, or implant.

In certain embodiments provided herein is a method of treating orameliorating a medical condition associated with premature terminationcodons (PTCs) in RNA. The method comprises administering to a subject acompound described herein or a composition described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the structure of(Z)-2-imino-5-((5-(2-nitrophenyl)furan-2-yl)methylene)thiazolidin-4-one,compound 5.

FIG. 2 provides a plots showing distribution of compound 5 in mdx miceafter systemic administration. Pharmacokinetics and biodistribution ofcompound 5 after intravenous (IV) and intraperitoneal (IP) injections.Injections were performed at a concentration of 5 mg/kg and of 30 mg/kgfor the IV and IP injection respectively. The predicted effective doseof the parent compound has been computed to be 2 mg/kg.

FIG. 3 provides a plot showing distribution of compound 5 in mdx miceafter systemic administration. Oral administration of compound 5 wasperformed at a dose of 60 mg/kg. The distribution of the compound wasassessed 60 min after oral gavage (OG) and compared to that achievedafter IV injection administered at a dose of 5 mg/kg or IP injectiongiven at a dose of 30 mg/kg.

FIG. 4 provides an exemplary chemical structure of prodrugs of compound5. The derivatives contain a linker (R1) which can be identical for allanalogs and a second group which can differs from prodrug to prodrug.The derivatives are designed to increase the solubility of the parentcompound and improve its absorption. Groups R2 through R7 represent 6possible structures that are conjugated to the linker to produce 6different prodrugs. The predicted site of cleavage in the liver is shownby the red arrow.

FIG. 5 provides a plot showing microsomal stability assay results inliver extracts. Compounds (5 μg) were incubated at 37° C. in fresh liverhomogenate (125 mg) isolated from C57 mice in the presence of NADPH (2mM).

FIG. 6 provides plots showing rate of conversion of prodrug into parentdrug (compound 5) in liver extract. Compounds (5 μg) were incubated at37° C. in fresh liver homogenate (125 mg) isolated from C57 mice in thepresence of NADPH (2 mM). Conversion of the prodrugs into the parentcompound was detected in all samples analyzed as early as 1 min afterincubation.

FIG. 7 provides plots showing dystrophin expression in mdx myotubes.Panel (A) provides a plot showing muscle cells isolated from mdx mousewhich were induced to differentiate for 16 hrs and then exposed to thecompounds for an additional 80 hrs prior to protein analysis. Dystrophinwas detected by Western blot analysis in all cells treated with theprodrugs but not in cells exposed to DMSO or in untreated mdx myotubes.As positive control, protein isolated from wild type myotubes maintainedin differentiation media for the same period of time (corresponding to5% of total protein) were mixed with proteins isolated from untreatedmdx. Equal loading was confirmed by Western blot using α-actinin asinternal control. Panel (B) provides plots showing quantitative analysisof protein levels obtained from multiple independent experiments andshowed a dose response increase in dystrophin expression in allcompounds tested. (†: significant to compound 5-2 μM; §: significant tocompound 5-10 μM; *≦0.03; **≦0.002)

FIG. 8 provides plots showing dystrophin expression after intramuscularinjection. Panel (A) provides plots showing dystrophin staining in TAmuscles of mdx mice injected with RTC13 or its derivatives and analyzedtwo weeks after delivery of compounds and compared to muscles treatedwith DMSO alone. Dystrophin expression was evident in muscles thatreceived the compounds but not in sham-injected muscles. Weak expressionwas detected in TA muscles injected with gentamicin and RTC14. (N=4muscles per compound; scale bar: 50 μm). Panel (B) provides plotsshowing the number of dystrophin positive fibers determined across thelength of the muscle. TA injected with compound 5 showed a significantlyhigher number of positive fibers than muscles that received compounds 16and 20. No significant differences were detected in muscles thatreceived compound 5 when compared to muscles that received compounds 12and 8. Shown here is the average number of dystrophin positive fibersper cross-sectional area containing the highest number of positives.(N=4 muscles per compound; *: p=0.0008; **: p≦0.04).

FIG. 9 provides a plot showing the rate of conversion of prodrugs intoparent drug (compound 5) in muscle in vivo. Prodrugs were injected intoTibialis Anterior and muscles were isolated 24 hrs after intramuscularinjection.

FIG. 10 provides a plot showing pharmacokinetic studies in mdx miceafter oral administration of compound 12 in liver and kidney. Thecompound was administered at a dose of 60 mg/kg in 6-8 weeks old C57BL/10 mice and tissues were analyzed after oral administration at thetime points indicated.

FIG. 11 provides plots showing the results of pharmacokinetics studiesin mdx mice after oral administration of compound 12 in muscle. Thecompound was administered at a dose of 60 mg/kg in 6-8 weeks old C57BL/10 mice and tissues were analyzed after oral administration at thetime points indicated.

FIG. 12 provides plots showing the results of pharmacokinetics studiesin mdx mice after oral administration of compound 8. The compound wasadministered at a dose of 60 mg/kg in 6-8 weeks old C57 BL/10 mice andtissues were analyzed 60 min after oral administration.

FIG. 13 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in plasma. Thecompound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in plasma 15 min, 30 min, 1 hr,4 hrs, 6 hrs, and 24 hrs after gavage.

FIG. 14 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in whole blood.The compound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in whole blood 15 min, 30 min, 1hr, 4 hrs, 6 hrs, and 24 hrs after gavage.

FIG. 15 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in liver. Thecompound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in liver 15 min, 30 min, 1 hr, 4hrs, 6 hrs and 24 hrs after gavage.

FIG. 16 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in kidney. Thecompound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in kidney 15 min, 30 min, 1 hr,4 hrs, 6 hrs and 24 hrs after gavage.

FIG. 17 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in quadriceps.The compound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in quadriceps 15 min, 30 min, 1hr, 4 hrs, 6 hrs, and 24 hrs after gavage.

FIG. 18 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in diaphragm.The compound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in diaphragm 15 min, 30 min, 1hr, 4 hrs, 6 hrs, and 24 hrs after gavage.

FIG. 19 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 ingastrocnemius. The compound was administered at the indicated dosages in6-8 weeks old C57BL/10 mice and tissues were analyzed in gastrocnemius15 min, 30 min, 1 hr, 4 hrs, 6 hrs, and 24 hrs after gavage. Compound 8and RTC13 (compound 5) were not detected in muscles isolated from micethat received compound 8 at a dose of 10 mg/kg.

FIG. 20 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in biceps. Thecompound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in biceps 15 min, 30 min, 1 hr,4 hrs, 6 hrs and 24 hrs after gavage. Compound 8 and RTC13 (compound 5)were not detected in muscles isolated from mice that received compound 8at a dose of 10 mg/kg.

FIG. 21 provides plots showing the results of pharmacokinetics studiesin C57BL/10 mice after oral administration of compound 8 in heart. Thecompound was administered at the indicated dosages in 6-8 weeks oldC57BL/10 mice and tissues were analyzed in heart 15 min, 30 min, 1 hr, 4hrs, 6 hrs, and 24 hrs after gavage. Compound 8 and RTC13 (compound 5)were not detected in muscles isolated from mice that received compound 8at a dose of 10 mg/kg and 30 mg/kg.

FIG. 22 provides plots showing the weight of mdx mice after oraladministration of compound 8 for up to one month. The compound wasadministered in 6-8 weeks old mdx and C57BL/10 mice three times per dayat the indicated dosages and weight was recorded daily.

FIG. 23 provides images of microscopic images obtained from musclesisolated from mdx mice after oral administration of compound 8.Compounds were administered in 6-8 weeks old mdx mice three times perday for one month at the indicated dosages. Dystrophin expression wasevident in muscles that received the compounds but not in sham-injectedmuscles. Muscles isolated from mice that received compound 8 at a doseof 30 mg/kg or 60 mg/kg showed higher levels of dystrophin expressionthan muscles obtained from mdx mice treated with PTC124 (N=6 muscles pertreatment group; scale bar: 100 μm).

FIG. 24 provides plots showing functional improvement in mdx mice afteroral administration of compound 8 for up to one month. Panel (A)provides plots showing the forelimb grip test that was used to assessthe effects of systemic administration of RTCs on muscle strength. Asignificant force recovery was detected in mdx mice treated withcompound 8 compared to sham-injected control mice and mice that receivedPTC124. Muscle strength remained significantly lower than that detectedin wild type mice. No significant improvements were detected in micethat received PTC124 compared to untreated mice. (N=5 mice per treatmentgroup; *p<0.05; **p<0.005). Panel (B) provides plots showing the resultsobtained using the wire test. The average hanging time of mice thatreceived compound 8 was significant than that of sham-injected mice ormice treated with PTC124. No significant improvements were detected inmice that received PTC124 compared to untreated mice. (N=6 mice pertreatment group; *p<0.05; **p=0.005).

FIG. 25 provides plots showing in vitro force measurement of diaphragmmuscles of mdx mice treated with vehicle, compound 8 at a dose of 30mg/kg or compound 8 at a dose of 60 mg/kg three times per day atintervals of 4 hrs for up to one month. Muscles isolated from untreatedand treated mice muscles were subjected to a series of five eccentriccontractions with a 5-min rest between contractions. Results werecompared to those of wild type (C57BL/10) mice. Panel (A) provides plotsshowing the decrease in force (in percentage) obtaining followingeccentric contractions over the course of the analyses. An improvementif loss of force was detected in mice that received compound 8 at a doseof 30 mg/kg and 60 mg/kg). Plot (B) show the results of the decrement inforce (in percentage) obtained in mice treated with compound 8. Thedecrement was significantly lower in mice that received compound 8 at adose of 60 mg/kg compared to mice that received vehicle only. (N=4 miceper treatment group, *:p≦0.05; **:p≦0.03).

FIG. 26 Table 2 provides the results of the serum chemistry analyses ofmdx mice treated with vehicle, PTC124 at a dose of 30 mg/kg, PTC124 at adose of 60 mg/kg, compound 8 at a dose of 30 mg/kg or compound 8 at adose of 60 mg/kg three times per day at intervals of 4 hrs for up to onemonth. The serum levels of albumin, alkaline phosphatase (ALP), totalbilirubin, lactate dehydrogenase (LDH), cholesterol, total protein, andglucose were within normal range. Mice that received compound 8 showed adecrease in blood urea nitrogen (BUN), alanine aminotransferase (ALT)and creatinine when compared to mice that received vehicle alone. Valuesare expressed as mean plus or minus Standard Deviation (±SD). Thesymbol * indicates statistical significance compared to mdx treated withvehicle only. The symbol † indicates statistical significance comparedto wild type (C57BL/10) untreated mice. (N=4 mice per treatment group;p≦0.05)

DETAILED DESCRIPTION Compounds

In an embodiment, provided herein is a compound which is a prodrug ofthe following compound 5 or 101:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.

In an embodiment, provided herein is a compound which is an acylderivative of the following compound 5 or 101:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.

In an embodiment, provided herein is a compound which is a prodrug ofthe following compound 5:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.

In an embodiment, provided herein is a compound which is an acylderivative of the following compound 5:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.

In an embodiment, provided herein is a compound which is a prodrug ofthe following compound 101:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.

In an embodiment, provided herein is a compound which is an acylderivative of the following compound 101:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.

In an embodiment, provided herein is compound according to Formula Ia:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, wherein: W is—NR^(a)R^(b), —C(O)OR⁴, —C(O)NR^(a)R^(b), or -HetAr; A is a bond fromC(O) to W, —(CH₂)_(f)CH(R¹)(CH₂)_(g)—,—(CH₂)_(f)C(R^(a)R^(b))(CH₂)_(g)—, —(CH₂CH₂O)_(h)(CH₂)_(t)—,—(CH₂)_(t)(OCH₂CH₂)_(h)—, —(CH₂)_(t)N(R^(e))CH₂CH₂Z, or

or, in the alternative, A and W combine to form

R¹ is —H, —(CH₂)_(n)CH₃, —CH(CH₃)CH₂CH₃, —CF₃, —CH₂(Ar), —CH₂(HetAr),—CH₂S(O)_(m)CH₃, —CH₂CH₂S(O)_(m)CH₃, —CH₂(CH₂)_(n)NR^(c)R^(d), —CH₂OH,—CH(CH₃)OH, or —(CH₂)_(t)COOH; R² is —H, —CH₃, —OH, or —CF₃; each ofR^(a) and R^(b) is independently R^(e); or, in the alternative, R^(a)and R^(b), together with the nitrogen or carbon atom to which they areattached, combine to form a 4 to 7-membered ring heterocyclic ring whichoptionally contains additional heteroatoms selected from O, NR^(g), andS(O)m; R^(c) is —H, —CH₃, —(CH₂)_(n)CH₃, —CH(R²)CH₃, —CH₂-pyridyl, orCH₂-imidazolyl; R^(d) is —H, —CH₃, or —(CH₂)_(n)CH₃; or, in thealternative, R^(c) and R^(d), together with the nitrogen atom to whichthey are attached, form a 4-7 membered heterocyclic ring whichoptionally contains additional heteroatoms selected from O, NR^(e), andS(O)_(m); each R^(e) is independently —H, —(CH₂)_(n)CH₃, —CH(CH₃)₂,—CH(CH₃)CH₂CH₃, —(CH₂CH₂O)_(p)R³, or CH₂HetAr; each R^(g) isindependently —H, —(CH₂)_(n)CH₃, —CH(CH₃)₂, —CH(CH₃)CH₂CH₃,—(CH₂CH₂O)_(p)R³, —CH₂-phenyl or —CH₂-phenyl optionally substituted withF, Cl, —CH₃, —OCH₃, —OCF₃, or HetAr; each R³ is independently —H, —CH₃,—OH, or —CF₃; or, alternatively, each R³ is independently —H, —CH₃,—CH₂CH₂—OH, or —CF₃; each R⁴ is independently —H or—(CH₂)_(n)NR^(c)R^(d); each HetAr is independently a heteroaryl groupoptionally selected from pyridyl, pyrimidyl, C-imidazolyl andN-imidazolyl; D is CH or N; Q is —O—, —NR^(a)—, —S(O)_(m)—, or —CHW—;each k is independently 1, 2, 3 or 4; each u is independently 1, 2 or 3;each v is independently 1, 2 or 3; each p is independently 1 or 2; eachf is independently 0, 1 or 2; each g is independently 0, 1 or 2; each his independently 1 or 2; each n is independently 0, 1, 2, 3, or 4; eachm is independently 0, 1 or 2; each of q₁ and q₂ is independently 0, 1, 2or 3; each s is independently 0, 1, 2 or 3; and each t is independently0, 1, 2 or 3. In certain embodiments, compounds according to Formula Iaare provided with the proviso that when D is CH, then Q is not —O—. Incertain embodiments, compounds according to Formula Ia are provided withthe proviso that when D is N and Q is —NR^(a)—, then u and v are notboth equal to 1. In certain embodiments, compounds according to FormulaIa are provided with the proviso that when D is N and Q is —NR^(a)—,—O—, or —S(O)_(m)—, then u and v are not both equal to 1. In embodimentsof Formula Ia when A is

and s and t are both 0, then A is

In an embodiment, provided herein is a compound of Formula Ia accordingto Formula Ib:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where A and W are asdescribed in the context of Formula Ia.

In an embodiment, provided herein is a compound of Formula Ia accordingto Formula II:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof and where R^(a) andR^(b) are as described in the context of Formula Ia.

In an embodiment, provided herein is a compound of Formula Ia accordingto Formula III:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where R^(a) and R^(b)are as described in the context of Formula Ia and wherein R^(x) is aside chain of a naturally occurring amino acid, or R^(x) is —CH₃, -iPr,—CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂COOH, —CH₂CH₂COOH,

In an embodiment, provided herein is compound of Formula Ia according toFormula IV:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where R^(a) and R^(b)are as described in the context of Formula Ia and wherein ring G is a 5to 6-membered heterocyclic ring. In an embodiment, provided herein is acompound according to Formula IV, wherein ring G is

In an embodiment, provided herein is a compound of Formula Ia accordingto Formula V:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, wherein Y is —OR⁴ or—NR^(a)R^(b) and where A, R⁴, R^(a) and R^(b) are as described in thecontext of Formula Ia.

In an embodiment, provided herein is a compound according to any ofFormulas I-V, wherein —NR^(a)R^(b) is —NH₂, —NHCH₃, —N(CH₃)₂,

In an embodiment, provided herein as a compound according to any ofFormulas I-V wherein —NR^(a)R^(b) is —NH₂, —NHCH₃, —N(CH₃)₂,

In an embodiment, provided herein is a compound according to any ofFormulas I-V, wherein A is —(CH₂)_(f)CH(R¹)(CH₂)_(g)—, where R¹, f and gare as described in the context of Formula Ia.

In an embodiment, provided herein is a compound of any of Formulas I-V,wherein W is HetAr.

In an embodiment, provided herein is a compound of any of Formulas I-V,wherein A is

where q₁, q₂, s and t are as described in the context of Formula Ia. Inan embodiment, provided herein is a compound of any of Formulas I-V,wherein A is —(CH₂CH₂O)_(h)(CH₂)_(t)— or —(CH₂)_(t)(OCH₂CH₂)_(h)—, and hand t are as described in the context of Formula Ia.

In an embodiment, provided herein is a compound of Formula Ia accordingto Formula VI:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where W is asdescribed in the context of Formula Ia.

In an embodiment, provided herein is a compound of any of Formulas I-VI,wherein A and W combine to form

where D, Q, k, u and v are as described in the context of Formula Ia.

In an embodiment, provided herein is a compound according to any of thefollowing formulas:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.

In certain embodiments, provided herein are:

(i) compounds as described herein, for example of Formula I-VI, 8, 12,16, 20 or 22, provided as a pharmaceutically acceptable salt; and

(ii) pharmaceutical compositions comprising: a compound as describedherein, for example of Formula I-VI, 8, 12, 16, 20 or 22, or apharmaceutically acceptable salt thereof, in an amount effective fortreating or ameliorating a medical condition associated with prematuretermination codons (PTCs) in RNA; and a pharmaceutically acceptableexcipient or pharmaceutically acceptable carrier.

In an embodiment, provided herein are compounds according to Formula Ia,wherein —C(O)-A-W combine to form:

In an embodiment, provided herein are compounds according to Formula I,wherein —C(O)-A-W combine to form:

In an embodiment, provided herein are compounds according to Formula I,wherein —C(O)-A-W combine to form:

In an embodiment, provided herein are compounds according to Formula I,wherein —C(O)-A-W combine to form:

In an embodiment, provided herein are compounds according to Formula I,wherein —C(O)-A-W combine to form:

Methods of Making

Compounds provided herein can be readily prepared according toestablished methodology in the art of organic synthesis. Several GeneralSynthetic Methods for preparation of compounds described herein areprovided.

Synthesis of the compounds with the Formula Ia are prepared essentiallyby acylation of the imino-thiazolidinone G2. The5-aryl/heteroaryl-2-imino-1,3-thiazolidin-4-one derivatives (G2) can beprepared by reaction of an appropriate aryl/heteroaryl aldehyde G3 with2-imino-1,3-thiazolidin-4-one (G4) as shown in scheme 1. Thearyl/heteroaryl aldehydes can be purchased from commercial sources orprepared following literature reports. Some of these reports include themethod described by Jung et al. (Bioorg. Med. Chem. Lett. 21 (2011)5842-5848); and some selective derivatives can be prepared as reportedby Chiacchio et al. (Tetrahedron, 59 (2003) 4733-4738) or as shown inSpecific Example 1 or alternatively by variations of these, and related,reported methods.

Reaction of an appropriately protected acid (G5), which is bearing abasic or an acidic functionality that would ultimately allow saltformation, with G2 using a variety of peptide coupling reagents providethe protected derivatives G6. The —COOH or the primary/secondary aminofunctionality in G5 are preferably protected with an acid labileprotecting group such as tert-butyl ester or BOC—, respectively. Anarray of coupling reagents are available commercially. Some of theseinclude a carbodiimide (EDCI, DCC etc.) with added coupling enhancerssuch as HOBt, HOAt or alternatively using TBTU, HBTU. Alternatively, thecarboxylic acid can be converted to an activated acyl derivative such asacid chloride, acid fluoride, or an active ester such as acylimidazolideor succinamide esters, prior to reaction with G2. If the acyl derivativeG6 contains an acid labile protecting group (e.g. —O-tert-butyl orN—BOC), treatment with an acid such as HCl in an organic solvent (MTBE,methyl-tert-butyl ether, THF, p-dioxane, EtOAC, or Et2O) provides thecorresponding hydrochloride salt of the amine or the free carboxylicacid containing compound G7. The amine-salt forms can be exchanged to analternate salt form or the —COOH can be converted to the carboxylatesalt form using an appropriate anion or cation ion-exchange resin,respectively.

Alternatively, the acid G5 can be converted to either a mixed anhydride(G8) or a symmetrical anhydride and subsequently reacted with the iminoderivative G2 to provide acylimino compounds G6 (Scheme 3).

The dicarboxylic acids (G10) can be converted to the anhydride (G11)which can be reacted directly with G2 to provide acylimino derivativesG12 (Scheme 4), and these can then be reacted with, for example, anethanolamine derivative to introduce a tertiary amine on to G12, ifdesired. Otherwise, the anhydride G11 can be converted to the mono-acidmonoester derivative G13, which can then be reacted with G2 as shown inscheme 2 or 3.

A wide variation of acids bearing diverse functionalities and structuralvariations at L1 are commercially available, or such desired inputs canbe prepared by numerous methods reported in the art. For example,reaction on a secondary amine (G14) with a halide G15/G19 (scheme 5 and6) or corresponding cycloalkyl/heterocyclic-keto-ester G22/G23 viareductive amination (scheme 5) would allow access to such acidderivatives. Subsequent hydrolysis of the ester (G16/G20/G24) providesthe corresponding acid or the carboxylate (G17/G21/G25). These can thenbe used as inputs for scheme 2/3. Moreover, the carboxylates itself canalso be used for acylation reactions, where the —COOH is generatedin-situ, as exemplified in Specific Examples 2 and 4.

Additional methods of synthesizing the compound can be found in, e.g.,Stuart Warren and Paul Wyatt, Workbook for Organic Synthesis: TheDisconnection Approach, second Edition, Wiley, 2010. Synthesis of thecompound is exemplified in the Examples where the preparation additionalcompounds is described in detail.

Methods of Use

In an embodiment, provided herein is a method of using the RTC. Themethod comprises applying the RTC to a subject an RTC described hereinto treat, prevent, or ameliorate a medical condition. The medicalcondition can be any disease or disorder caused by or otherwiseassociated with PTC.

In some embodiments, the method can be conducted in living bodies ofmammals. In such a case, the compounds can be administered to themammals. In one embodiment, the mammals can be patients with geneticdiseases caused by nonsense mutation, and the method can be conducted asa treatment method of genetic diseases caused by nonsense mutation.

In an embodiment, provided herein are:

(i) methods of treating a disease or disorder in a subject caused by orassociated with one or more premature termination codons of the subject,the method comprising administering a therapeutically effective amountof a compound or composition as described herein, for example of FormulaI-VI, 8, 12, 16, 20 or 22, or prodrug or acyl derivative of compound 5or 101, to the subject;

(ii) a method according to (i), wherein the disease or disorder isselected from the group consisting of central nervous system diseases,ataxia-telangiectasia, muscular dystrophy, Duchenne muscular dystrophy(DMD), Dravet syndrome, myotonic dystrophy, multiple sclerosis,infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachsdisease, neural tissue degeneration, Parkinson's disease, autoimmunediseases, chronic rheumatoid arthritis, lupus erythematosus,graft-versus-host disease, primary immunodeficiencies, severe combinedimmunodeficiency, DNA Ligase IV deficiency, DNA repair disorders,Nijmegen breakage disorders, xeroderma pigmentosum (XP), inflammatorydiseases, rheumatoid arthritis, blood diseases, hemophilia, vonWillebrand disease, thalassemia, familial erythrocytosis,nephrolithiasis, collagen diseases, osteogenesis imperfecta, cirrhosis,neurofibroma, bullous disease, lysosomal storage disease, Hurler'sdisease, familial cholesterolemia, cerebellar ataxia, tuberoussclerosis, immune deficiency, kidney disease, lung disease, cysticfibrosis, familial hypercholesterolemia, pigmentary retinopathy,amyloidosis, atherosclerosis, gigantism, dwarfism, hypothyroidism,hyperthyroidism, aging, obesity, diabetes mellitus, Niemann-Pickdisease, Marfan syndrome, neuromuscular diseases, Becker musculardystrophy (BMD), spinal muscular atrophy, cancer, and any geneticdisorder caused by nonsense mutation(s);

(iii) a method of (i) or (ii), wherein the disease or disorder isassociated with a premature stop codon in an ATM or dystrophin gene ofthe subject;

(iv) a method according to (iii), where the disease or disorder isselected from ataxia-telangiectasia, breast cancer, and lymphoreticularmalignancies;

(v) a method for enhancing production in a subject of a functionalprotein from a gene disrupted by the presence of a premature stop codonin the coding region of the gene, comprising administering to thesubject a compound or composition described herein, for example acompound of Formula I-VI, 8, 12, 16, 20 or 22, or prodrug or acylderivative of compound 5 or 101, in an amount effective to suppress thepremature stop codon and increase transcription of the gene, optionallywherein the gene is an ATM or dystrophin gene; and

(vi) a method for enhancing production in a subject of a functionalprotein, where production of the protein is disrupted by a geneticmutation, comprising administering to the subject a compound orcomposition described herein, for example a compound of Formula I-VI, 8,12, 16, 20 or 22, or prodrug or acyl derivative of compound 5 or 101, inan amount effective to suppress the genetic mutation and/or correct adefect caused by the mutation and to increase transcription of the gene,optionally wherein the gene is an ATM or dystrophin gene.

As used herein, the term disorder and medical condition can be usedinterchangeably and generally refer to a disease attributable to aninternal termination codon in a gene (a premature termination codon)generated by such as a point mutation, deletion, and insertion in thegene which leads to inhibition of expression of protein having a normalfunction, or attributable to degradation of mRNA that contains thepremature termination codon which leads to inhibition of proteinexpression. The genetic disease caused by nonsense mutation is notspecifically limited, but is exemplified by the following: centralnervous system diseases such as muscular dystrophy, Duchenne musculardystrophy, multiple sclerosis, infantile neuronal ceroid lipofuscinosis,Alzheimer's disease, Tay-Sachs disease, neural tissue degeneration, andParkinson's disease; autoimmune diseases such as chronic rheumatoidarthritis and graft-versus-host disease; inflammatory diseases such asarthritis; blood diseases such as hemophilia, von Willebrand disease,ataxia telangiectasia, thalassemia, familial erythrocytosis, andnephrolithiasis; collagen diseases such as osteogenesis imperfecta andcirrhosis; neurofibroma; bullous disease; lysosomal storage disease;Hurler's disease; familial cholesterolemia; cerebellar ataxia; tuberoussclerosis; immune deficiency; kidney disease; lung disease; cysticfibrosis; familial hypercholesterolemia; pigmentary retinopathy;amyloidosis; atheroscleroses; gigantism; dwarfism; hypothyroidism;hyperthyroidism; aging; obesity; diabetes mellitus; Niemann-Pickdisease; Marfan syndrome; and cancer. The term “cancer”, such as cancerassociated with a nonsense mutation of a suppressor gene such as p53gene, includes all types of cancer, which is exemplified by lung cancer,colon and rectal cancer, stomach cancer, esophagus cancer, kidneycancer, pancreatic cancer, prostate cancer, breast cancer, uteruscancer, ovary cancer, skin cancer and brain tumor.

Pharmaceutical Compositions

In another aspect, a pharmaceutical composition for use in treatment orprevention of the genetic diseases caused by nonsense mutation isprovided, wherein the pharmaceutical composition comprises as aneffective ingredient a compound expressed by any one of theaforementioned formulae a pharmacologically acceptable salt or prodrugthereof

The pharmaceutical composition preferably comprises a compound describedabove or a pharmacologically acceptable salt or prodrug thereof.

The pharmaceutical composition more preferably comprises a compoundshown in the aforementioned table.

In the aforementioned aspect, the pharmaceutical composition can containa pharmacologically acceptable carrier or excipients. An amount of thecompound used in the pharmaceutical composition is not limited as far asit is an effective amount for treatment. The genetic disease caused bynonsense mutation is not specifically limited, but is exemplified by thefollowing: central nervous system diseases such as muscular dystrophy,Duchenne muscular dystrophy, multiple sclerosis, infantile neuronalceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neuraltissue degeneration, and Parkinson's disease; autoimmune diseases suchas chronic rheumatoid arthritis and graft-versus-host disease;inflammatory diseases such as arthritis; blood diseases such ashemophilia, von Willebrand disease, ataxia telangiectasia, thalassemia,familial erythrocytosis, and nephrolithiasis; collagen diseases such asosteogenesis imperfecta and cirrhosis; neurofibroma; bullous disease;lysosomal storage disease; Hurler's disease; familial cholesterolemia;cerebellar ataxia; tuberous sclerosis; immune deficiency; kidneydisease; lung disease; cystic fibrosis; familial hypercholesterolemia;pigmentary retinopathy; amyloidosis; atherosclerosis; gigantism;dwarfism; hypothyroidism; hyperthyroidism; aging; obesity; diabetesmellitus; Niemann-Pick disease; Marfan syndrome; and cancer. The term“cancer”, such as cancer associated with a nonsense mutation of asuppressor gene such as p53 gene, includes all types of cancer, which isexemplified by lung cancer, colon and rectal cancer, stomach cancer,esophagus cancer, kidney cancer, pancreatic cancer, prostate cancer,breast cancer, uterus cancer, ovary cancer, skin cancer and brain tumor.

The pharmaceutical composition in the aspect can contain, as activeingredients, the aforementioned compound and other compounds, or cancontain a mixture of two or more aforementioned compounds.

The pharmacologically acceptable salt in the present specification isnot specifically limited as far as it can be used in medicaments.Examples of a salt that the compounds described herein forms with a baseinclude the following: salts thereof with inorganic bases such assodium, potassium, magnesium, calcium, and aluminum; salts thereof withorganic bases such as methylamine, ethylamine and ethanolamine; saltsthereof with basic amino acids such as lysine and ornithine; andammonium salt. The salts can be acid addition salts, which arespecifically exemplified by acid addition salts with the following:mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, nitric acid, and phosphoric acid:organic acids suchas formic acid, acetic acid, propionic acid, oxalic acid, malonic acid,succinic acid, fumaric acid, maleic acid, lactic acid, malic acid,tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonicacid; acidic amino acids such as aspartic acid and glutamic acid.

Further, the compounds described herein include hydrates thereof,various pharmaceutically acceptable solvates thereof, and polymorphiccrystals thereof.

The pharmaceutical compositions described herein can be formulated invarious dosage forms, which are exemplified by the following: oraladministration forms such as tablets, capsules, powders, granules,pills, liquids, emulsions, suspensions, solutions, spirits, syrups,extracts, and elixirs; parenteral administration forms such asinjections, for example, subcutaneous injections, intravenousinjections, intramuscular injections, and intraperitoneal injections;transdermal administration forms, plasters and pressure sensitiveadhesives, ointments or lotions; intramouth administration forms such assublingual forms and oral patch preparations; and nasal administrationforms such as aerosols, but are not limited thereto. These preparationscan be manufactured by using a known method generally used in a drugmanufacturing process. In one embodiment, the pharmaceutical compositiondescribed herein can be administered for treating muscular disease as aninjection such as an intramuscular injection for administering directlyinto muscle.

The pharmaceutical compositions can contain various kind of ingredientsgenerally used, for example, one or more pharmaceutically acceptablefillers, disintegrators, diluents, lubricants, flavoring agents,colorants, sweetening agents, corrigents, suspending agents, humectants,emulsifying agents, dispersing agents, auxiliary agents, preservatives,buffers, binders, stabilizers, and coating agents. In addition, thepharmaceutical composition described herein can be sustained-releasedosage forms or extended-release dosage forms.

Dosage ranges of the pharmaceutical compositions are not particularlylimited, and can be determined in accordance with the following:effectiveness of the ingredients contained therein; the administrationform; the route of administration; the type of disease; thecharacteristics of the subject (e.g., body weight, age, symptomaticconditions, and whether a subject is taking other pharmaceuticalagents); and the judgment of a physician in charge. In general, asuitable dosage can fall, for example, within a range of about 0.01 μgto 100 mg, per 1 kg of the body weight of the subject, and preferablywithin a range of about 0.1 μg to 1 mg, per 1 kg of body weight.However, the dosage can be altered using conventional experiments foroptimization of a dosage that are well-known in the art. Theaforementioned dosage can be divided for administration once to severaltimes a day. Alternatively, periodic administration once every few daysor few weeks can be employed.

The pharmaceutical compositions can be administered to a patient whosebiological sample obtained in advance is subjected to a study forpresence or absence of premature termination codons in genes containedtherein and is found to have a detected premature termination codon. Abiological sample can be any ones insofar as it contains nucleic acids,and is exemplified by cells, bloods, cerebrospinal fluids,bronchoalveolar lavage fluids, expectorations, or other body fluids aswell as biopsy tissues. Nucleic acid samples can be prepared from thebiological samples for use. The nucleic acid samples can be prepared bywell-known nucleic acid preparation methods. The nucleic acid samplescan be DNA or RNA. The nucleic acid samples prepared can be useddirectly for detection, or can be subjected to enzymatic amplificationof predetermined region thereof by PCR or other amplification methods inadvance for analysis. Detection of a termination codon can be carriedout by using well-known methods for detecting genetic mutations such asDNA sequencing, Southern blot, polymerase chain reaction (PCR), shorttandem repeat (STR), or restricted fragment length polymorphism. Thedetection method is not limited to the exemplified methods, and anymethod can be used insofar as it can detect a premature terminationcodon. Alternatively, the presence of a premature termination codon canbe detected by measuring an amount of mRNA derived from thepredetermined gene in the biological sample and detecting reduction ofthe amount of the mRNA compared to an amount of mRNA derived from thegene in a biological sample obtained from healthy subject. mRNA can bemeasures by using known analysis methods such as northern blotting.

In terms of a route of administration of the pharmaceutical composition,it can be either systemic administration or local administration. Theroute of administration that is appropriate for a particular disease,symptomatic condition, or other factors, should be selected. Forexample, parenteral administration including normal intravenousinjection, intra-arterial administration, subcutaneous administration,intracutaneous administration, and intramuscular administration can beemployed. Oral administration can be also employed. Further,transmucosal administration or transdermal administration can beemployed.

The term “read-through” herein means to skip over a prematuretermination codon in ribosomal translation, or to substitute an aminoacid, or to suppress degradation of mRNA that comprises a prematuretermination codon.

In the aforementioned aspect, a sequence that comprises a prematuretermination codon derive from responsible genes for diseases caused bynonsense mutation is not specifically limited insofar as it is asequence comprising a termination codon such as TAA, TAG, or TGA, in areading flame. The sequence is preferably around 20 to 150 by long. Inone embodiment, the sequence can be a sequence containing a sequencethat comprises a premature termination codon of humans or animals havinggenetic disease caused by nonsense mutation including animal models forthe diseases. For example, such a gene can contain a prematuretermination codon in the dystrophin gene of mdx mice.

Preferably the composition is adapted for oral administration, e.g. inthe form of a tablet, coated tablet, dragee, hard or soft gelatincapsule, solution, emulsion or suspension. In general the oralcomposition will comprise from 1 mg to 400 mg of such agent. It isconvenient for the subject to swallow one or two tablets, coatedtablets, dragées, or gelatin capsules per day. However, the compositioncan also be adapted for administration by any other conventional meansof systemic administration including rectally, e.g. in the form ofsuppositories, parenterally, e.g. in the form of injection solutions, ornasally.

The biologically active compounds can be processed with pharmaceuticallyinert, inorganic or organic carriers for the production ofpharmaceutical compositions. Lactose, corn starch, or derivativesthereof, talc, stearic acid or its salts and the like can be used, forexample, as such carriers for tablets, coated tablets, dragees and hardgelatin capsules. Suitable carriers for soft gelatin capsules are, forexample, vegetable oils, waxes, fats, semi-solid and liquid polyols andthe like. Depending on the nature of the active ingredient no carriersare, however, usually required in the case of soft gelatin capsules,other than the soft gelatin itself. Suitable carriers for the productionof solutions and syrups are, for example, water, polyols, glycerol,vegetable oils and the like. Suitable carriers for suppositories are,for example, natural or hardened oils, waxes, fats, semi-liquid orliquid polyols and the like.

The pharmaceutical compositions can, moreover, contain preservatives,solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners,colorants, flavorants, salts for varying the osmotic pressure, buffers,coating agents or antioxidants. They can also contain still othertherapeutically valuable substances, particularly antidiabetic orhypolipidemic agents that act through mechanisms other than thoseunderlying the effects of the compounds described herein. Agents whichcan advantageously be combined with compounds described herein in asingle formulation include but are not limited to biguanides such asmetformin, insulin releasing agents such as the sulfonylurea insulinreleaser glyburide and other sulfonylurea insulin releasers,cholesterol-lowering drugs such as the “statin” HMG-CoA reductaseinhibitors such as atrovastatin, lovastatin, pravastatin andsimvastatin, PPAR-alpha agonists such as clofibrate and gemfibrozil,PPAR-gamma agonists such as thiazolidinediones (e.g. rosiglitazone andpioglitazone, alpha-glucosidase inhibitors such as acarbose (whichinhibit starch digestion), and prandial insulin releasers such asrepaglinide. The amounts of complementary agents combined with compoundsdescribed herein in single formulations are in accord with the dosesused in standard clinical practice. Established safe and effective doseranges for certain representative compounds are set forth above.

The invention is described in more detail in the following illustrativeexamples. Although the examples can represent selected embodimentsdescribed herein, it is intended that the following examples areillustrative and not limiting.

EXAMPLES Example 1 Comparative Preparation of(5Z)-2-imino-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-1,3-thiazolidin-4-one(compound 5)

Step-1: 5-(2-nitrophenyl)furan-2-carbaldehyde (3). To a stirred solutionof 2-nitro aniline (100 g, 0.72 mol) in 6 N HCl (1000 mL) was heated upto 80° C. and stirred for 30 min. The solution was cooled to 0° C. and asolution of sodium nitrite (55 g, 0.796 mol in 180 mL of water was addeddrop wise over 30 min. To the ice cold solution of the diazonium saltwas added 2-furfuraldehyde (70 g, 0.724 moles) over 30 min, followed bydrop wise addition of solution of cupric chloride (17 g, 0.1264 mol in180 mL water) over 20 min at 0° C. Reaction mass was stirred at roomtemperature for 30 h. Solid was filtered and wet cake was stirred in 1:1dioxane/ethanol (150 mL) for 15 min, solid was filtered, washed withhexane (100 mL) and air dried to provide 55 g of (3) as a light yellowsolid. (Yield: 35%). M.P:98-100° C. ¹H-NMR (CDCl3): ppm 9.71 (1H, s),7.86 (1H, m), 7.04 (1H, m), 7.67 (1H, m), 7.35, (1H, m), 7.34 (1H, s),6.81 (1H, m).

Step-2:(5Z)-2-imino-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-1,3-thiazolidin-4-one(5) [RTC-13]. To a stirred solution of aldehyde (50 g, 0.23 mol) inacetic acid (1500 mL) was added 2-imino-1,3-thiazolidin-4-one (27 g,0.23 mol) and sodium acetate (75 g, 0.924 mol) was heated to 100° C. for18 h. Reaction mass at 70° C. was filtered (undissolved solid wasdiscarded), filtrate was cooled to room temperature and 500 mL of waterwas added. The resulting solid was filtered and washed with water (500mL). The wet cake was dried at 70° C. for 8 h under reduced pressure.The above solid was refluxed in toluene (600 mL) for 1 h, filtered whilehot and the solid compound was dried in a vacuum oven at 60° C. underreduced pressure (520 mmHg) to provide 27 g (yellow solid), isolatedyield: 38%. HPLC=98% purity (area percent, 254 nm). M.P: 263-265° C.¹H-NMR (DMSO-d6): ppm 9.4 (1H, s), 9.2 (1H, s), 8.0 (1H, d, 6 Hz), 7.9(1H, d, 6 Hz), 7.8 (1H, t, 6 Hz), 7.65 (1H, t, 6H), 7.45 (1H, s), 7.11(2H, m). LC-MS: 316 (MH+, ES+APCI, positive mode). Elemental Analysis:calculated for Cl4H9N3O4S, Element: Expt. (Theory)=C: 53.08 (53.33), H:3.00 (2.88), N: 12.96 (13.33) and S: 10.26 (10.17).

Example 2 Preparation of prodrug derivative2-(4-methylpiperazin-1-yl)-N-[(2E,5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]acetamidedihydrochloride (8)

Step-1:2-(4-methylpiperazin-1-yl)-N-[(2E,5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]acetamide(7). To a stirred solution of sodium (4-methylpiperazin-1-yl)acetate(0.50 g, 2.77 mmol) in dichloromethane (15 mL) was added EDCI.HCl (0.6g, 3.04 mmol), HOBt (0.4 g, 3.04 mmol) and DIPEA (0.386 g, 3.04 mmol) at0° C., mixture was stirred at 0° C. for 30 min. A solution of RTC-13(0.8 g, 0.00253 mol) in 15 mL DMA was added at 0° C. Reaction mixturewas stirred at room temperature for 5 h. Reaction mass was diluted withMTBE and water (1:1 v/v, 200 mL), the turbid mass was filtered, thelayers were separated, aq. layer was extracted with 10% MeOH/CHCl₃ (50ml×2), the organic (CHCl₃) layer was dried over anhydrous sodiumsulphate, and evaporated to provide 0.6 g of a yellow solid as free-base(7) (Yield: 54%). HPLC=93% purity (area percent, 254 nm). HPLC-93%.LC-MS: 456 (MH+, ES+APCI, positive mode).

Step-2: A suspension of (7) (0.20 g, 0.439 mmol) in acetonitrile (6 mL)was cooled to 0° C. added 15% HCl in MTBE (158 mg, 4.329 mmol) andstirred at same temperature for 1 h. The solid was filtered and washedwith hexane (20 mL) to provide, after drying 0.20 g of (8) as a yellowsolid. (Yield: 87%). HPLC (area percent, 254 nm) 97% purity.M.P:198-202° C. ¹H-NMR: (DMSO-d6) ppm 11.5 (0.6H, bs), 8.15 (1H, d, 6Hz), 7.92 (1H, d, 6 Hz), 7.85 (1H, t, 6 Hz), 7.75 (1H, t, 6 Hz), 7.70(1H, s), 7.35 (1H, d, 3 Hz), 7.20 (1H, d, 3 Hz), 4.10 (2H, bs), 3.2-3.6(8H, m), 2.8 (3H, s). Elemental Analysis calculated forC21H21N505S.2HCl.2H2O, Element: Expt (Theory)=C: 44.69 (44.09), H: 4.82(4.86), N: 12.41 (12.28), S: 5.68 (5.93).

Example 3 Preparation of prodrug derivative:2-(4-methylpiperazin-1-yl)-N-[(2E,5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]acetamidedihydrochloride (8)

Step-1:2-(4-methylpiperazin-1-yl)-N-[(2E,5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]acetamide(7). To a stirred solution of (4-methyl-1-piperzinnyl)acetic acid (6.62g, 0.03809 mol) in dichloromethane (100 ml) was added EDCI.HCl (7.35 g,0.03809 mol), HOBT (5.82 g, 0.03809 mol) and DIPEA (11.2 ml, 0.063 mol)at 0° C., stirred at same temperature for 30 mints and at roomtemperature for 30 mint. A solution of (5) (10 g, 0.03174 mol in 1100 mlDMA, dissolved at 60° C., and then cooled to room temperature) drop wisein 20 mints at 0° C. Further mixture was stirred at room temperature for24 h. Solid was separated out from the mixture, filtered and washed withdichloromethane (25 ml). The obtained compound (8 g) was stirred inwater (80 ml) for 1 h and filtered, dried at 50° C. to get a constantweight (7 g). The dry compound was stirred in methanol (200 mL) for 1 hand filtered, dried under high vacuum pump at room temperature to get aconstant weight, to provide 6.7 g of a yellow color solid, the free-base(7). Yield: 47%. HPLC=98% purity (area percent, 254 nm). ¹HNMR: matchedwith spectra from example 2, step-1. LC-MS: 465 (MH+, ES+APCI, positivemode). Elemental Analysis: calculated for C21H21N5O5S, Element: Expt.(Theory)=C: 54.83 (55.37), H: 5.12 (4.65), N: 15.10 (15.38) and S: 6.74(7.04).

Step-2: Salt formation. A suspension of (7) (0.20 g, 0.439 mmol) inacetonitrile (6 mL) was cooled to 0° C., 15% HCl in MTBE (158 mg, 4.329mmol) was added and stirred at 0° C. for 1 h. The solid was filtered andwashed with hexane (20 mL), dried under high vacuum to provide 0.20 g of(8) as a yellow solid, (yield 87%). HPLC (area percent, 254 nm) 97%purity. M.P:198-202° C. ¹H-NMR: (DMSO-d6) ppm 11.5 (0.6H, bs), 8.15 (1H,d, 6 Hz), 7.92 (1H, d, 6 Hz), 7.85 (1H, t, 6 Hz), 7.75 (1H, t, 6 Hz),7.70 (1H, s), 7.35 (1H, d, 3 Hz), 7.20 (1H, d, 3 Hz), 4.10 (2H, bs),3.2-3.6 (8H, m), 2.8 (3H, s). ¹H-NMR: (TFA-d6) ppm 8.22 (1H, bs), 8.15(1H, m), 7.65 (2H, bs), 7.58 (1H, m), 7.50 (1H, d, 3 Hz), 7.18 (1H, d, 3Hz), 5.05 (2H, bs), 4.40 and 4.30 (8H, two overlapping broad singlets),3.30 (3H, s). Elemental Analysis calculated for C21H21N5O5S.2HCl,Element: Expt (Theory) C: 47.97 (47.73), H: 4.92 (4.39), N: 13.24(13.25), and S: 6.05 (6.07).

Step-2: Salt formation. To a stirred suspension of (7) (6.2 g, 13.6mmol) in acetonitrile (185 mL) was cooled to 0° C., 25% MTBE.HCl(tert-butylmethylether.HCl) (3.4 g (14 ml), 95 mmol) was added drop wisein 20 min and stirred at 0° C. for 2 h. The solid was filtered and thewet cake was triturated with 200 mL of methanol, sonicated for 5 min andfiltered, solid was dried under high vacuum at room temperature to aconstant weight to provide 6.1 g of (8) as a yellow color solid, (Yield:80%). HPLC=99.0% purity (area percent, 254 nm). M.P:198-202° C. ¹HNMR:consistent with data shown for example 2 and 3, step-2. ElementalAnalysis calculated for C21H21N5O5S-2HCl-1.5H2O: Element: Expt(Theory)=C: 45.41 (45.53), H: 4.72 (5.02), N: 12.61 (12.51), S: 5.77(5.87).

Example 4 Preparation of prodrug derivative:(E)-N²,N²-dimethyl-N-[(5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]glycinamidehydrochloride (12)

Step-1:(E)-N²,N²-dimethyl-N-[(5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]glycinamide(11). A solution of sodium salt (0.70 g, 5.6 mmol) in THF (20 mL) wascooled to 0° C., isobutyl chloroformate (0.76 g, 5.7 mmol) was added,followed by N-methylmorpholine (1.14 g, 11.4 mmol), reaction mixturestirred at same temperature for 1 h. A solution of (5) (1.2 g, 3.8 mmol)in dimethylacetamide (15 mL) was added at 0° C. over 5 min. The reactionmixture was stirred at 0° C. for 6 h. Reaction mass was diluted withMTBE and water (1:1 v/v, 200 mL), and the solid was filtered. The solidobtained solid (600 mg) was purified by silica gel column eluting with20% acetone/CHCl₃ to provide 0.3 g of a yellow solid (11). (Yield: 20%).¹HNMR (DMSO-d6) ppm 8.00 (1H, d, 6 Hz), 7.93 (1H, d, 6 Hz), 7.85 (1H, t,6 Hz), 7.65 (1H, t, 6 Hz), 7.35 (1H, s), 7.1 (2H, s), 3.8 (2H, s),3.2-3.5 (bs), 2.65 (6H, s).

Setp-2: Salt formation. A suspension of 9× (0.180 g, 0.45 mmol) inacetonitrile (6 mL) was cooled to 0° C., 1 mL of 15% MTBE.HCl (162 mg,4.5 mmol) was added and stirred at same temperature for 1 h. Theresulting compound was filtered and washed with hexane (20 mL), driedunder vacuo to provide 0.20 g of (12) as a light greenish solid. (Yield:98%). HPLC=99.0% purity (area percent, 254 nm). LC-MS: 401 (MH+,ES+APCI, positive mode). ¹HNMR (DMSO-d6) ppm 8.07 (1H, d, 6 Hz), 7.95(1H, d, 6 Hz), 7.85 (1H, t, 6 Hz), 7.75 (1H, t, 6 Hz), 7.70 (1H, s),7.35 (1H, d, 3 Hz), 7.25 (2H, d, 3 Hz), 4.4 (2H, s), 3.35 (1H, bs), 2.9(6H, s).

Example 5 Preparation of prodrug derivative:N-[(2E,5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]piperidine-4-carboxamidehydrochloride (16)

Step-1: To a solution of 1-(tert-butoxycarbonyl)piperidine-4-carboxylicacid (5 g, 21.8 m mol) in a mixture of dichloromethane and water (60mL:80 mL) was added chloromethyl chlorosulfonate (4.3 g, 26 mmol) dropwise, subsequently solid NaHCO₃ (7 g, 84 mmol) was added portion wise,followed by n-tetrabutylammonium hydrogen sulphate (TBAHS) (0.7 g, 2.1mmol) at room temperature. The resulting reaction mixture was stirred atroom temperature for 5 h. The organic layer was separated, washed withwater and dried over anhydrous sodium sulfate, filtered and concentratedin vacuo to provide 5.2 g of a white solid, yield: 86%. This wasconfirmed by LCMS and used as such for next step.

Step-2: To a solution of (5) (2 g, 6.3 mmol) in DMA (25 mL) was addedcesium carbonate (2.45 g, 7.5 mmol) at room temperature, stirred for 15min, and then cooled to 0° C., followed by addition of (14) (2.25 g, 8.1mmol). The reaction mixture was stirred at room temperature for 5 h.Reaction mass was diluted with MTBE and water (1:1 v/v, 100 mL). Layerswere separated and aq. layer was extracted with 10% MeOH/CHCl₃ (50mL×2). The combined organic layer was dried over anhydrous sodiumsulphate, filtered and concentrated in vacuo to provide 0.4 g of (15) ayellow solid. This was the only product observed during the reaction andwas isolated. ¹HNMR (DMSO-d6) ppm 12.5 (0.8H, bs), 8.05 (1H, d, 6 Hz),7.90 (1H, d, 6 Hz), 7.85 (1H, t, 6 Hz), 7.70 (1H, t, 6 Hz), 7.62 (1H,s), 7.25 (1H, d, 3 Hz), 7.15 (2H, d, 3 Hz), 3.95 (1H, m), 2.7-2.9 (4H,m), 1.85 (2H, m), 1.45 (2H, m), 1.35 (9H, s).

Step-3: To a suspension of (15) (0.4 g, 0.76 mmol) in MTBE (20 mL) 15%MTBE.HCl (1.8 mL, 0.27 g, 15 mmol) was added at 0-5° C. and stirred atsame temperature for 24 h, still starting material was observed, furtheradded 1.8 mL of MTBE.HCl (0.27 g, 0.015 mol) and stirred for additional18 h. Reaction mass was diluted with ACN (15 mL) and filtered. The solidwas stirred in hexane (20 mL) for 10 min and filtered to provide 0.208 gof a light yellow solid, (yield: 48%). MP: 290-293° C. HPLC=95.8% purity(area percent, 254 nm). LC-MS: 427 (MH+, ES+APCI, positive mode). ¹HNMR(DMSO-d6) ppm 9.0 (1H, bs), 8.75 (1H, bs), 8.05 (1H, d, 6 Hz), 7.90 (1H,d, 6 Hz), 7.85 (1H, t, 6 Hz), 7.73 (1H, t, 6 Hz), 7.65 (1H, s), 7.35(1H, d, 3 Hz), 7.18 (2H, d, 3 Hz), 3.3 (1H, m), 2.8-3.0 (4H, m), 2.02(2H, m), 1.80 (2H, m).

Example 6 Preparation of prodrug derivative:2-(morpholin-4-yl)-N-[(2E,5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]acetamide(20)

Step-1: To a solution of (5) (3 g, 9.5 mmol) in THF (250 mL) DMAP (1.5g, 1.2 mmol) was added at −5° C., and chloromethyl chloroformate (1.2mL, 11.8 mmol) drop wise at 0-5° C. and further stirred at 0° C. for 12h. Reaction mass was diluted with water (700 mL) and filtered, thecompound was dried at rt for 5 h to provide 3 g of a pale yellow solid(LC/MS confirmed) and was used as such for the next step. To a 1 g (2.45mmol) solution in acetonitrile (100 mL) was added sodiummorpholin-4-ylacetate (18) (0.82 g, 4.9 mmol) followed by TBAHS (1.2 g,3.5 mmol) at room temperature and stirred at ambient temperature for 5h. HPLC of the reaction mixture indicated a new product and smallamounts of the starting (5). Reaction mass was filtered and the solidwas stirred in water and filtered, the compound was dried at 50° C. for5 h under vacuo to provide 0.38 g of (19) as a yellow solid, (yield35%). ¹HNMR (DMSO-d6) ppm 8.05 (1H, d, 6 Hz), 7.92 (1H, d, 6 Hz), 7.85(1H, t, 6 Hz), 7.70 (1H, t, 6 Hz), 7.65 (1H, s), 7.30 (1H, d, 3 Hz),7.15 (1H, d, 3 Hz), 3.6 (4H, m), 3.45 (2H, s), 2.6 (4H, m).

Step-3: A suspension of (19) (0.150 g, 0.339 mmol) in acetonitrile (5mL) was cooled to 0° C., 15% MTBE.HCl (0.8 ml, 122 mg, 3.34 mmol) wasadded and stirred at same temperature for 30 min. The compound wasfiltered, washed with hexane (20 ml) and dried under vacuo to provide0.110 g of (20) as a yellow solid, (yield: 68%). HPLC=95.3% purity (areapercent, 254 nm). LC-MS: 443 (MH+, ES+APCI, positive mode). M.Pt.:226-230° C. 1HNMR (DMSO-d6) ppm 8.1 (1H, d, 6 Hz), 7.95 (1H, d, 6 Hz),7.90 (1H, 7, 6 Hz), 7.5 (2H, m), 7.4 (1H, d, 3 Hz), 7.25 (1H, d, 3 Hz),4.45 (2H, s), 3.90 (4H, bs), 3.2-3.5 (4H, bs). ¹H-NMR: (TFA-d6) ppm 8.15(1H, s), 8.10 (1H, d, 6 Hz), 7.80 (2H, s), 7.75 (1H, m), 7.50 (1H, d, 3Hz), 7.10 (1H, d, 3 Hz), 4.80 (2H, bs), 4.40 (4H, bs), 3.80 (2H, m),3.65 (2H, m).

Example 7 Preparation of derivative tert-butyl[(2S)-1-{[(2E,5Z)-5-{[5-(2-nitrophenyl)furan-2-yl]methylidene}-4-oxo-1,3-thiazolidin-2-ylidene]amino}-1-oxopropan-2-yl]carbamate(22)

To a stirred solution of BOC-L-alanine (21) (59 mg, 0.32 mmoles) indichloromethane (5 mL) and diisopropylethylamine (61 mg, 0.47 mmol) at0-5° C. were added EDCI.HCl (73 mg, 0.378 mmol) and HOBt (51 mg, 0.37mmol). The resulting reaction mixture was stirred at 0-5° C. for 30 min.A solution of (5) (100 mg, 0.317 mmol) in DMA (5 mL) was added and themixture was stirred overnight at room temperature. The completion of thereaction was monitored by TLC. The reaction mixture was poured intoice-cold water (25 mL) and extracted with dichloromethane (3×20 mL). Thecombined organic layer was washed with water and dried over anhydrousNa2SO4, filtered and concentrated under reduced pressure, to provide 45mg of (22) as a yellow solid. ¹HNMR (CDCl3) ppm 12.0 (0.85H, bs), 7.90(1H, d, 6 Hz), 7.55 (1H, d, 6 Hz), 7.75 (2H, m), 7.55 (1H, t, 6 Hz), 7.0(1H, d, 3 Hz), 6.85 (1H, d, 3 Hz), 6.35 (0.7H, bs), 4.65 (1H, m), 1.6(3H, d), 1.35 (9H, s).

Example 8 Aqueous Solubility

The solubility of selected compounds was tested and compared to that ofcomparative compound 5. To 5 mg of each test compound, water was slowlyadded initially in 0.5 mL and then 0.1 mL increments with vortexing forapproximately 1 minute in between each aliquot of water addition andvisually inspected for homogeneity. Comparative compound 5 was dilutedto 1 L with water and vigorously stirred for up to 12 hr, but still thesample did not appear to dissolve fully. Table 1 provides the aqueoussolubility of various prodrugs.

TABLE 1 Aqueous Solubility of Selected Compounds Solubility in (mg/mL)water at Compound No. ambient pH, at room temperature 5 <0.005(Comparative)  8 3.3 12 10.0 16 0.18 20 0.1

While particular embodiments described herein have been shown anddescribed, it will be obvious to those skilled in the art that changesand modifications can be made without departing from this invention inits broader aspects. Therefore, the appended claims are to encompasswithin their scope all such changes and modifications as fall within thetrue spirit and scope of this invention.

What is claimed is:
 1. A compound which is a prodrug of the followingcompound 5 or 101:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.
 2. A compound which isan acyl derivative of the following compound 5 or 101:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.
 3. A compoundaccording to Formula Ia:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, wherein: W is—NR^(a)R^(b), —C(O)OR⁴, —C(O)NR^(a)R^(b), or -HetAr; A is a bond fromC(O) to W, —(CH₂)_(f)CH(R¹)(CH₂)_(g)—,—(CH₂)_(f)C(R^(a)R^(b))(CH₂)_(g)—, —(CH₂CH₂O)_(h)(CH₂)_(t)—,—(CH₂)_(t)(OCH₂CH₂)_(h)—, —(CH₂)_(t)N(R^(e))CH₂CH₂Z, or

or, in the alternative, A and W combine to form

R¹ is —H, —(CH₂)_(n)CH₃, —CH(CH₃)CH₂CH₃, —CF₃, —CH₂(Ar), —CH₂(HetAr),—CH₂S(O)_(m)CH₃, —CH₂CH₂S(O)_(m)CH₃, —CH₂(CH₂)_(n)NR^(c)R^(d), —CH₂OH,—CH(CH₃)OH, or —(CH₂)_(t)COOH; R² is —H, —CH₃, —OH, or —CF₃; each ofR^(a) and R^(b) is independently R^(e); or, in the alternative, R^(a)and R^(b), together with the nitrogen or carbon atom to which they areattached, combine to form a 4 to 7-membered ring heterocyclic ring whichoptionally contains additional heteroatoms selected from O, NR^(g), andS(O)m; R^(c) is —H, —CH₃, —(CH₂)_(n)CH₃, —CH(R²)CH₃, —CH₂-pyridyl, orCH₂-imidazolyl; R^(d) is —H, —CH₃, or —(CH₂)_(n)CH₃; or, in thealternative, R^(c) and R^(d), together with the nitrogen atom to whichthey are attached, form a 4-7 membered heterocyclic ring whichoptionally contains additional heteroatoms selected from O, NR^(e), andS(O)_(m); each R^(e) is independently —H, —(CH₂)_(n)CH₃, —CH(CH₃)₂,—CH(CH₃)CH₂CH₃, —(CH₂CH₂O)_(p)R³, or CH₂HetAr; each R^(g) isindependently —H, —(CH₂)_(n)CH₃, —CH(CH₃)₂, —CH(CH₃)CH₂CH₃,—(CH₂CH₂O)_(p)R³, —CH₂-phenyl or —CH₂-phenyl optionally substituted withF, Cl, —CH₃, —OCH₃, —OCF₃, or HetAr; each R³ is independently —H, —CH₃,—CH₂CH₂—OH, or —CF₃; each R⁴ is independently —H or —(CH₂)—NR^(c)R^(d);each HetAr is independently a heteroaryl group optionally selected frompyridyl, pyrimidyl, C-imidazolyl and N-imidazolyl; D is CH or N; Q is—O—, —NR^(a)—, —S(O)_(m)—, or —CHW—; each k is independently 1, 2, 3 or4; each u is independently 1, 2 or 3; each v is independently 1, 2 or 3;each p is independently 1 or 2; each f is independently 0, 1 or 2; eachg is independently 0, 1 or 2; each h is independently 1 or 2; each n isindependently 0, 1, 2, 3, or 4; each m is independently 0, 1 or 2; eachof q₁ and q₂ is independently 0, 1, 2 or 3; each s is independently 0,1, 2 or 3; and each t is independently 0, 1, 2 or
 3. 4. The compound ofany of the above claims, with the proviso that when D is N and Q is—NR^(a)—, —O—, or —S(O)m-, then u and v are not both equal to
 1. 5. Thecompound of any of the above claims according to Formula Ib:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.
 6. The compound any ofthe above claims according to Formula II:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof and where R^(a) andR^(b) are as described in the context of Formula Ia.
 7. The compound ofany of the above claims according to Formula III:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where R^(a) and R^(b)are as described in the context of Formula Ia and wherein R^(x) is aside chain of a naturally occurring amino acid or R^(x) is —CH₃, -iPr,—CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂COOH, —CH₂CH₂COOH,


8. The compound of claim 7, wherein R^(x) is —CH₃, -iPr, —CH₂NH₂,—CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂COOH, —CH₂CH₂COOH,


9. The compound of any of the above claims according to Formula IV:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where R^(a) and R^(b)are as described in the context of Formula Ia and wherein ring G is a 5to 6-membered heterocyclic ring.
 10. The compound of claim 9, whereinring G is


11. The compound of any of the above claims according to Formula V:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where A is asdescribed in the context of Formula Ia and wherein Y is —OR⁴ or—NR^(a)R^(b).
 12. The compound of any of the above claims, wherein—NR^(a)R^(b) is —NH₂, —NHCH₃, N(CH₃)₂,


13. The compound of any of the above claims, wherein A is—(CH₂)_(f)CH(R¹)(CH₂)_(g)—, where R¹, f and g are as described in thecontext of Formula Ia.
 14. The compound of any of the above claims,wherein W is HetAr.
 15. The compound of any of the above claims, whereinA is

where q₁, q₂, s and t are as described in the context of Formula Ia. 16.The compound of any of the above claims, wherein A is—(CH₂CH₂O)_(h)(CH₂)_(t)— or —(CH₂)_(t)(OCH₂CH₂)_(h)—, and h and t are asdescribed in the context of Formula Ia.
 17. The compound of any of theabove claims according to Formula VI:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where W is asdescribed in the context of Formula Ia.
 18. The compound of any of theabove claims, wherein A and W combine to form

where D, Q, k, u and v are as described in the context of Formula Ia.19. The compound of any of claims 1-3 according to any of the followingformulas:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof.
 20. The compound ofany of claims 3-19 which is a pharmaceutically acceptable salt.
 21. Apharmaceutical composition comprising: a compound, or a pharmaceuticallyacceptable salt thereof, in an amount effective for treating orameliorating a medical condition associated with premature terminationcodons (PTCs) in RNA, wherein the compound is according to any of claims1-20; and a pharmaceutically acceptable excipient or pharmaceuticallyacceptable carrier.
 22. A method of treating a disease or disorder in asubject caused by or associated with one or more premature terminationcodons of the subject, the method comprising administering atherapeutically effective amount of a compound or composition accordingto any of claims 1-21 to the subject.
 23. The method of claim 22,wherein the disease or disorder is selected from the group consisting ofcentral nervous system diseases, ataxia-telangiectasia, musculardystrophy, Duchenne muscular dystrophy, Dravet syndrome, myotonicdystrophy, multiple sclerosis, infantile neuronal ceroid lipofuscinosis,Alzheimer's disease, Tay-Sachs disease, neural tissue degeneration,Parkinson's disease, autoimmune diseases, chronic rheumatoid arthritis,lupus erythematosus, graft-versus-host disease, primaryimmunodeficiencies, severe combined immunodeficiency, DNA Ligase IVdeficiency, DNA repair disorders, Nijmegen breakage disorders, xerodermapigmentosum (XP), inflammatory diseases, rheumatoid arthritis, blooddiseases, hemophilia, von Willebrand disease, thalassemia, familialerythrocytosis, nephrolithiasis, collagen diseases, osteogenesisimperfecta, cirrhosis, neurofibroma, bullous disease, lysosomal storagedisease, Hurler's disease, familial cholesterolemia, cerebellar ataxia,tuberous sclerosis, immune deficiency, kidney disease, lung disease,cystic fibrosis, familial hypercholesterolemia, pigmentary retinopathy,amyloidosis, atherosclerosis, gigantism, dwarfism, hypothyroidism,hyperthyroidism, aging, obesity, diabetes mellitus, Niemann-Pickdisease, Marfan syndrome, neuromuscular diseases, Becker musculardystrophy (BMD), spinal muscular atrophy, cancer, and any geneticdisorder caused by nonsense mutation(s).
 24. The method of claim 22 or23 wherein the disease or disorder is associated with a premature stopcodon in an ATM or dystrophin gene of the subject.
 25. The method ofclaim 24 where the disease or disorder is selected from Duchennemuscular dystrophy (DMD), ataxia-telangiectasia, breast cancer, andlymphoreticular malignancies.
 26. A method for enhancing production in asubject of a functional protein from a gene disrupted by the presence ofa premature stop codon in the coding region of the gene, comprisingadministering to the subject a compound or composition according to anyof claims 1-19 in an amount effective to suppress the premature stopcodon and increase transcription of the gene.
 27. The method of claim 26wherein the gene is an ATM or dystrophin gene.
 28. A method forenhancing production in a subject of a functional protein, whereproduction of the protein is disrupted by a genetic mutation, comprisingadministering to the subject a compound or composition according to anyof claims 1-19 in an amount effective to suppress the genetic mutationand/or correct a defect caused by the mutation and to increasetranscription of the gene.
 29. The method of claim 28 wherein the geneis ATM.
 30. A method for the preparation of a compound of Formula Ia:

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, where A, W and R areas described in the context of Formula Ia; according to Schemes 1-7 orany of Examples 2-7.
 31. A compound having the ability to read throughpremature termination codons (PTCs) in RNA, of Formula (9001):

or a pharmaceutically acceptable salt, solvate, polymorph, hydrate,ester, isomer, stereoisomer, or tautomer thereof, wherein: W═NRaRb,COOH, HetAr; A=A1, A2, A3, or A4; A1=—(CH₂)_(f)—CH(R1R2)-(CH₂)_(g)—;R1=H, (CH₂)_(n)CH₃, CH(CH₃)CH₂CH₃, CF₃, CH(Ar), CH(HetAr),CH₂S(O)_(m)CH₃, CH₂CH₂S(O)_(m)CH₃, CH₂(CH₂)_(n)—NRcRd, CH₂OH, CH(CH₃)OH,(CH₂)_(t)—COOH; Rc=H, CH₃, (CH₂)_(n)CH₃, CH(R₂)CH₃, CH₂-pyridyl,CH₂—Imidazolyl; Rd=H, (CH₂)_(n)CH₃; or Rc and Rd taken together form a4-7 membered ring which can optionally contain heteroatoms such as O,NRe, S(O)_(m); Re═H, (CH₂)_(n)CH₃, CH(CH₃)₂, CH(CH₃)CH₂CH₃,(CH₂CH₂O)_(p)R2, CH₂-HetAr; f=0-2; g=0-2; n=0-4; p=1-2; m=0-2; R2=H,CH₃, OH, CF₃, or R1 and R2 taken together can form a 4-7 membered ringwhich can optionally contain O, NRe, S(O)_(m); Re═H, (CH₂)_(n)CH₃,CH(CH₃)₂, CH(CH₃)CH₂CH₃, (CH₂CH₂O)_(p)R2; optionally carbocycliccompounds selected from cyclobutane, cyclopentane, cyclohexane, andcycloheptane; optionally oxygen containing rings selected from oxetane,tetrahydrofurane, dioxane, and oxepine; optionally nitrogen containingrings selected from azetidine, pyrrolidine, piperidine, and azepine;optionally sulfur containing rings selected from thioxatane,tetrahydrothiofurane, and corresponding sulfoxides or sulphones thereof;or wherein —CO-A-W of Formula 9001 is of Formula 9002:

where Z═O, N—Re, S(O)₀₋₂ A2=

q1 or q2=0-3; s=0-3; t=1-3; A3=—(CH₂CH₂—O)_(h)—(CH₂)_(t)—,—(CH₂)_(t)—(O—CH₂CH₂)_(h)—, —(CH₂)_(t)—N(Re)—CH₂CH₂—Z; h=1-2; A4: when‘A-W’ taken together

P═CH, N; Q=O, NRa, S(O)_(m), or CH—W; k=1-4; u=1-3; v=1-3; with theproviso that when P═CH, Q cannot be ═O and u and v each cannot be =1when P═N and Q=NRa; A5 A can be absent, and —CO and W are connected by abond; Ra or Rb can independently be Re; or Ra and Rb taken together forma 4-7 membered ring which can optionally contain a O, NRg, S(O)_(m);Rg=H, (CH₂)_(n)CH₃, CH(CH₃)₂, CH(CH₃)CH₂CH₃, (CH₂CH₂O)_(p)R2,(CH₂)phenyl or (CH₂)phenyl optionally substituted with F, Cl, CH₃, OCH₃,OCF₃, or heteroaromatics, HetAr=pyridyl, pyrimidyl, C-Imidazolyl,N-imidazolyl; when the A and/or W contains—carboxylic acidfunctionality, appropriate salt forms (sodium, potassium, ammonium,quaternary ammonium (e.g. Me₄N⁺), and other pharmaceutical acceptablesalt form are included; and when the A and/or W contains an animefunctionality, in particular a tertiary amine or a basic heteroarylfunctionality, the corresponding salt form such as hydrochloride,hydrobromide, phosphate, toluene sulphonate, methyl sulphonate, acetate,maleate, fumarate, and other pharmaceutically acceptable salt forms areincluded.
 32. A composition, comprising at least one compound or apharmaceutically acceptable salt or prodrug thereof, including basicand/or acidic functionalities in an amount effective for treating orameliorating a medical condition associated with premature terminationcodons (PTCs) in RNA, wherein the compounds are according to claim 31.33. The composition of claim 32, comprising two compounds, wherein eachof the two compounds is according to claim
 31. 34. The composition ofclaim 32, further comprising a pharmaceutically acceptable carrier. 35.The composition of claim 32, further comprising a stable isotope (D,13C, etc.) and/or radioisotope (¹⁸F, ¹¹C, etc.).
 36. The compounds ofclaim 32 which are crystalline or amorphous.
 37. The composition ofclaim 32, in a formulation for local or systemic delivery administeredas aqueous solution or which can or cannot be buffered.
 38. Thecomposition of claim 32, in a formulation for local or systemic deliveryformulated with additional commonly accepted excipient(s) andadministered to mammals as solution, suspension, microemulsion,microsuspension, nanosuspension, or as a solid dosage form.
 39. Thecomposition of claim 32, in a formulation for oral administration,injection, topical administration, pulmonary administration, or implant.40. A compound or composition described herein which is administered tohumans in dose ranging from 1 microgram to 10 grams.
 41. The compositionof claim 32, wherein the medical condition is selected from the groupconsisting of Duchenne muscular dystrophy (DMD), central nervous systemdiseases, an autoimmune diseases; inflammatory diseases; blood diseases;collagen diseases; neurofibroma; bullous disease; lysosomal storagedisease; Hurler's disease; familial cholesterolemia; cerebellar ataxia;tuberous sclerosis; immune deficiency; kidney disease; lung disease;cystic fibrosis; familial hypercholesterolemia; pigmentary retinopathy;amyloidosis; atheroscleroses; gigantism; dwarfism; hypothyroidism;hyperthyroidism; aging; obesity; diabetes mellitus; Niemann-Pickdisease; Marfan syndrome; and cancer.
 42. A method of treating orameliorating a medical condition associated with premature terminationcodons (PTCs) in RNA, comprising administering to a subject a compoundor composition according to any of the above claims.
 43. The method ofany of the above claims, wherein the medical condition is selected fromthe group consisting of Duchenne muscular dystrophy (DMD), centralnervous system diseases, an autoimmune diseases; inflammatory diseases;blood diseases; collagen diseases; neurofibroma; bullous disease;lysosomal storage disease; Hurler's disease; familial cholesterolemia;cerebellar ataxia; Choroideremia (CHM); tuberous sclerosis; immunedeficiency; kidney disease; lung disease; cystic fibrosis; familialhypercholesterolemia; pigmentary retinopathy; amyloidosis;atheroscleroses; gigantism; dwarfism; hypothyroidism; hyperthyroidism;aging; obesity; diabetes mellitus; Niemann-Pick disease; Marfansyndrome; and cancer.